Effects of Yacon (Smallanthus sonchifolius) Juice Byproduct Administered Using Different Feeding Methods on the Growth Performance, Digestive Enzyme Activity, Antioxidant Status, and Disease Resistance against Streptococcus iniae of Juvenile Black Rockfish (Sebastes schlegelii)

Abstract

This study was designed to evaluate the effects of yacon (Smallanthus sonchifolius) juice byproduct (YJB) on the growth performance, digestive and antioxidant enzyme activities, and disease resistance against Streptococcus iniae of juvenile black rockfish (Sebastes schlegelii) based on different feeding schedules. Four different YJB feeding strategies were evaluated: feeding the fish a basal diet continuously (control, T0), feeding them YJB (2.5 g/kg) continuously (T1), feeding them YJB for 1 day and the basal diet the next day (T2), and feeding them YJB for 1 day and the basal diet for the following 2 days (T3). No difference in survival among the treatments was found after the 8-week feeding trial (p > 0.05). However, the T1 and T2 groups exhibited significant enhancements in final body weight, weight gain, and specific growth rate compared with the T0 and T3 groups. Furthermore, the T1 and T2 groups showed a significant improvement in feed consumption, feed efficiency, and protein efficiency ratio compared with the T0 and T3 groups. No significant differences in the condition factor or viscerosomatic and hepatosomatic indices were observed among all the groups. Intestinal amylase, trypsin, and lipase activity was significantly (p < 0.05) higher in the T1 and T2 groups than in the T0 and T3 groups. Lysozyme, superoxide dismutase, and catalase activity along with glutathione peroxidase content were significantly (p < 0.05) higher under all YJB feeding regimens than those under the control treatment. The survival rates in all the YJB treatment groups after the S. iniae challenge were significantly (p < 0.05) enhanced. In conclusion, we recommend offering YJB at day-to-day intervals to improve growth performance, digestive enzyme activity, antioxidant status, and disease resistance against S. iniae.

1. Introduction

The provision of functional feed additives is becoming an increasingly common alternative method for increasing aquaculture production and preventing disease [1,2,3,4,5]. In particular, phytogenic nutraceutical-based feeds are regarded as important for fish farming due to their capacity to function in enhancing growth performance, feed digestion, immunity, and resistance to biotic and abiotic stresses [6,7,8,9,10]. Plant-based feed additives (essential oils, plant extracts, and plant food industry byproducts) are superior to other additives owing to their high levels of organic substances, which have no impact on fish and human health and do not disrupt the environment or ecology [11,12].

Plant byproducts, a plant-based feed additive, are mainly derived from the vegetables and fruits used to manufacture products such as juice, jam, and canned food [13]. Byproducts from the agricultural industry have garnered considerable attention because they pose a constant threat as environmental contaminants and are a significant operational issue for the food sector [12]. Nonetheless, byproducts from the agricultural industry, such as the pomaces of apples, tomatoes, citrus, and grapes, might be a source of potentially useful substances that could be utilized as functional supplements in animal feed throughout the food chain [12,14,15,16]. The use of plant byproducts in tending organic livestock has recently been allowed [17]. In aquatic animal culture, the above feeding approach converts byproducts or waste from plant food processing into high-value seafood and contributes to the lowering of the feed-to-food competition ratio in the aquatic animal production chain, an effect that has important technical consequences for the entire food chain. Several studies have investigated the possibility of using plant-based food-processing byproducts in aquafeeds [6,18,19]. Frosi et al. [20] reported that plant byproducts from food processing contain several active nutrients, such as polyphenols, flavonoids, tannins, anthocyanins, pigments, essential oils, minerals, fatty acids, and bioactive peptides. These bioactive chemicals contribute to a variety of pharmacological actions that help activate the antioxidative, immunological, and antistress responses of aquatic animals [13,21]. Plant byproducts from food processing are also abundant in polysaccharides, which are considered prebiotic supplements necessary for enhancing intestinal microbial balance, digestibility, and local intestinal immunity [22,23].

Yacon (Smallanthus sonchifolius) is a plant with tuberous roots that is native to the Andes and widely distributed in South America [24]. It is recognized as a functional food primarily because of its fructan components (fructo-oligosaccharides [FOSs] and inulin), phenolic compounds, and flavonoids [25]. The advantages of yacon have attracted attention worldwide [26] because consumers are increasingly becoming interested in foods that provide health benefits beyond nutrition. In yacon-producing countries, yacon roots are used to make a variety of products, including flour, dehydrated products, vegetable slices or chips, tea (made from dry leaves), juices, purees, and sweeteners [26]. In Korea, yacon is usually either consumed fresh or processed into juice [27]. A substantial amount of pomace is produced during the production of yacon juice. Oh et al. [28] reported that yacon juice byproduct (YJB) contains FOSs, phenolics, and flavonoids. Because of these components, the incorporation of 2.5 g/kg of YJB in black rockfish (Sebastes schlegelii) feed improved growth performance, digestive activity, antioxidant status, and nonspecific immunity [28]. Lee et al. [29] also showed that YJB improved growth performance, antioxidant capacity, and disease resistance against Vibrio anguillarum when used as a functional phytofeed additive in the diet of S. schlegelii.

S. schlegelii is considered a commercially important cultured marine species in East Asian countries, including China, Japan, and Korea [30,31]. Intensive culture systems, which are common in S. schlegelii farming, frequently generate stressful conditions that reduce fish growth and well-being. Feeding costs account for approximately two-thirds of the overall cost of fish farming worldwide. The application of excessive feed additives in addition to the expenses of regular production decreases the profitability of fish culture. Furthermore, the continuous application of functional feed additives, such as phytoadditives, probiotics, and prebiotics, increases the expenses of the fish-culturing process, and finding a balance between the positive benefits and the desired outcome is highly recommended [1,32,33]. Amphan et al. [32] reported that feeding Nile tilapia (Oreochromis niloticus) every other 2 weeks or every 4 days is the optimal choice for β-glucan administration. Dawood et al. [1] stated that administering Aspergillus oryzae every 2 days enhanced the growth of fish, and they advised supplying this supplement at daily intervals to enhance the growth performance, digestive enzyme activity, intestinal histomorphology, and blood health of O. niloticus. Developing an optimal feeding strategy with low cost and high efficiency based on the type of additive, target fish species, and required dietary dose is necessary. Also, in Korea, feeding costs account for approximately two-thirds of the overall cost of fish farming. The application of excessive feed additives in addition to the expense of regular production decreases the profitability of fish culture. Hence, examining the efficiency of feed additive administration via various feeding strategies is of increasing importance. However, the effects of phytoadditive feeding frequency on the performance of S. schlegelii are unknown. Thus, this study aims to investigate the effect of YJB feeding regimes on the growth performance, intestinal digestive enzyme activity, antioxidant enzyme activity, and Streptococcus iniae resistance of S. schlegelii.

2. Materials and Methods

2.1. Fish and Experimental Diets

Juvenile S. schlegelii specimens were obtained from a commercial hatchery (Namhae-gun, Gyeongsangnam-do, Republic of Korea). The present experiment was carried out at the Marine Bio-Education and Research Center, Gyeongsang National University (Tongyeong, Gyeongsangnam-do, Republic of Korea). The fish were kept for 2 weeks in a 1.5 ton round polyethylene tank and fed commercial extruded pellets (Jeil Feed Co., Haman, Gyeongsangnam-do, Republic of Korea; 52% crude protein and 10% crude lipids) prior to the feeding trial. Water quality parameters were monitored daily throughout the adaptation. Water temperature, dissolved oxygen, and salinity were measured by using a YSI-Pro20 (YSI Inc., Yellow Springs, OH, USA). The average rearing water characteristics during the adaptation were as follows: a water temperature of 22.1 ± 0.60 °C, a dissolved oxygen level of 7.7 ± 0.28 mg/L, and a salinity of 31.11 ± 0.38 psu. After the completion of acclimatization, 480 juvenile rockfish with an initial average weight of 15.5 g were randomly assigned to 12 tanks (water: 250 L) with 3 replicates per group (40 fish per tank). The tanks had a water flow rate of 2.7 L/min.

A basal diet was formulated (

Table 1. Experimental diet formulation (g/kg, dry matter basis).

	Experimental Diets
	Basal Diet	YJB
Jack mackerel meal	520	520
Fermented soybean meal	120	120
Wheat flour	255	252.5
YJB a	0	2.5
Fish oil	40	40
Soybean oil	40	40
Vitamin premix b	10	10
Mineral premix c	10	10
Choline	5	5
Proximate composition (g/kg)
Dry matter	932	932
Crude protein	478	480
Crude lipids	133	132
Ash	91	96

^a YJB (yacon juice byproduct) supplied by Youngjin Health Food Store (Daegu, Republic of Korea).^b The vitamin premix contained the following ingredients diluted in cellulose (given in g/kg mix): L-ascorbic acid, 121.2; DL-α-tocopheryl acetate, 18.8; thiamin hydrochloride, 2.7; riboflavin, 9.1; pyridoxine hydrochloride, 1.8; niacin, 36.4; Ca-D-pantothenate, 12.7; myo-inositol, 181.8; D-biotin, 0.27; folic acid, 0.68; p-aminobenzoic acid, 18.2; menadione, 1.8; retinyl acetate, 0.73; cholecalciferol, 0.003; cyanocobalamin, 0.003. ^c The mineral premix contained the following ingredients (given in g/kg mix): MgSO₄·7H₂O, 80.0; NaH₂PO₄·2H₂O, 370.0; KCl, 130.0; ferric citrate, 40.0; ZnSO₄·7H₂O, 20.0; Ca-lactate, 356.5; CuCl, 0.2; AlCl₃·6H₂O, 0.15; KI, 0.15; Na₂Se₂O₃, 0.01; MnSO₄·H₂O, 2.0; CoCl₂·6H₂O, 1.0.

). The YJB diet was supplemented with dry YJB powder (Youngjin Health Food Store, Daegu, Republic of Korea) at a dose of 2.5 g/kg (Table 1). The dose of YJB was set in reference to studies on juvenile rockfish [28]. All dry ingredients were mechanically well-combined into a homogenous mixture to formulate the experimental diets. Then, fish and soybean oils and distilled water were added into the mixture to achieve a uniform texture appropriate for pelleting (3.0–4.0 mm pellets) by using a chopper (3.0 mm diameter, SL Machinery, Incheon, Republic of Korea). The obtained pellets were then dried at 20 °C in an agricultural product dryer (KED-M07D1, Kiturami Co., Ltd., Seoul, Republic of Korea) for 48 h. Then, the experimental feeds were stored at −20 °C until use.

2.2. Feeding Trial

Two sets of experimental diets (the basal and YJB-supplemented diets) were formulated. Four groups in which different feeding strategies were applied were designed: the first group was fed the basal diet continuously (T0), the second group was fed the YJB diet continuously (T1), the third group was fed the YJB diet for 1 day and the basal diet the next day (T2), and the fourth group was fed the YJB diet for 1 day and the basal diet for the next 2 days (T3) for 8 weeks. The fish were hand-fed twice a day at 08:00 and 17:00. Fecal matter was removed, and the amount of feed consumed by the fish in each tank was recorded daily. Water quality parameters were monitored daily throughout the feeding trial. Water temperature, dissolved oxygen, and salinity were measured by using a YSI-Pro20 (YSI Inc., Yellow Springs, OH, USA). The average rearing water characteristics during the trial were as follows: a water temperature of 22.9 ± 0.90 °C, a dissolved oxygen level of 7.6 ± 0.34 mg/L, and a salinity of 31.49 ± 0.24 psu.

2.3. Growth Performance and Feed Utilization

The lengths and weights of all the fish in each tank were individually measured at the end of the feeding trial. For this purpose, all the fish were fasted for 24 h and anesthetized using 150 ppm of tricaine methanesulfonate MS-222 (Sigma-Aldrich, Saint Louis, MO, USA). Finally, growth performance and feed utilization were calculated in accordance with the following formulas:

Survival (SR, %) = (number of fish at the end of the trial/number of fish at the beginning of the trial) × 100,

Weight gain (WG) = final body weight − initial body weight,

Specific growth rate (SGR, %/day) = [ln final weight of fish – ln initial weight of fish/days of feeding] × 100,

Feed consumption (FC, g/fish) = total dry feed intake/number of surviving fish,

Feed efficiency (FE) = WG of fish/feed consumed,

Protein efficiency ratio (PER) = WG of fish/amount of protein consumed,

Condition factor (CF) = Fish weight × 100/total length³.

2.4. Proximate Whole-Body Composition

To analyze chemical compositions, a homogenized paste was prepared by finely chopping and processing the whole bodies of 5 fish sampled from each tank. The Kjeldahl digestion method was used to determine the crude protein (N × 6.25) content using a KD310–A–1015 KjelROC Analyzer (OPSIS Liquid LINE, Furulund, Sweden). The Soxhlet extraction method was used to evaluate the crude lipid content using a Sox-tec extractor (ST 243 Soxtec™; FOSS, Hillerod, Denmark). The moisture and ash proportions were analyzed via oven drying at a temperature of 105 °C for 24 h and by using a muffle furnace at a temperature of 600 °C for 4 h, respectively.

2.5. Plasma Biochemical Indices and Antioxidant Enzyme Activities

Blood samples were taken from the caudal veins of 5 fish per tank (15 fish per group) using a heparin-coated syringe and centrifuged in a microtube centrifuge at 2000× g for 10 min. Plasma samples were collected and stored at −80 °C until the biochemical assays and antioxidant enzyme analysis were conducted. An automatic chemistry system (Fuji Dri-Chem NX500i; Fujifilm, Tokyo, Japan) was used to determine different plasma biochemical parameters, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (T-CHO), total protein (TP), and glucose (GLU) levels. The activities of superoxide dismutase (SOD) and catalase (CAT) and the concentrations of glutathione (GSH) in the plasma samples were determined using a commercial kit (Cayman’s Assay Kit, Cayman Chemical, Ann Arbor, MI, USA) in accordance with the manual’s instructions. Absorbance was measured using a spectrophotometer (Thermo Scientific MULTISKAN GO, Vantaa, Finland).

2.6. Lysozyme Activities

For serum collection, blood was collected from the caudal veins of another 3 fish per tank using syringes without anticoagulant and allowed to clot for 30 min, centrifuged in a microtube centrifuge at 3000× g for 5 min, and then stored at −80 °C for lysozyme activity analysis. A turbidimetric assay was performed in accordance with the method reported by Lange et al. [34] to measure serum lysozyme activity. In short, lysozyme activity was measured by adding 100 μL of test blood to a 1.9 mL suspension of Micrococcus lysodeikticus (0.2 mg/mL; Sigma, St. Louis, MO, USA) in a 0.05 M sodium phosphate buffer (pH 6.2). The reactions took place at 25 °C, and a spectrophotometer (Thermo Fisher Scientific, Tewksbury, MA, USA) was used to measure the absorbance at 530 nm between 0 and 60 min. As the lysozyme activity unit, the amount of enzyme required to generate a 0.001/min reduction in absorbance was considered.

2.7. Digestive Enzyme Measurements

The intestines of 5 fish per tank were dissected to obtain samples. These samples were then homogenized in a solution of ice-cold 0.86% physiological saline at a ratio of 10 volumes to weight. The homogenization process was carried out in an ice bath using a TissueLyser II (QIAGEN, Venlo, Netherlands). The resulting mixture was then centrifuged at 13,000 rpm for 10 min at 4 °C to obtain the supernatant. The enzymatic activities of amylase, trypsin, and lipase were assessed using a commercially available kit (Abcam, Cambridge, UK), following the guidelines stated in the supplementary manual.

2.8. S.iniae Challenge

Twenty fish were randomly selected from each tank to conduct a challenge test. The chosen fish were subsequently redistributed into the tanks. The S. iniae strain used was obtained from the Korean Culture Collection of Aquatic Microorganisms of the National Institute of Fisheries Science (Busan, Republic of Korea). The fish were subjected to artificial infection through intraperitoneal injection using a 0.1 mL pathogenic S. iniae culture suspension with a concentration of 5.0 × 10⁶ CFU/mL. The water temperature was consistently held at 20.5 ± 0.15 °C (mean ± SE), and the concentrations of dissolved oxygen were maintained at 7.1 ± 0.24 mg/L. The daily survival rate of fish was documented over 12 days post-infection. Fish mortality was recorded at 12 h intervals throughout the observation period.

2.9. Statistical Analysis

The data were presented as means ± standard errors. To remove the variance heterogeneity in survival percent values, they were arcsine-transformed before being subjected to one-way analysis of variance. The homogeneity of variances among the treatments was tested using Levene’s test. One-way analysis of variance and Tukey’s HSD multiple-range tests were used to assess the mean differences among various groups, with a significance level of p < 0.05. Fish survival during the 12-day post-observation period after artificial S. iniae injection was analyzed using Kaplan–Meier survival curves and Log-rank and Wilcoxon tests. The statistical analyses were conducted using SPSS version 27.0 (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Growth Performance

The growth and feed utilization indices of juvenile rockfish subjected to different feeding strategies involving the dietary supplementation of YJB are shown in

Table 2. Growth performance of juvenile rockfish under different feeding regimens incorporating the YJB-supplemented diet.

	Experimental Diets				p-Value
	T0	T1	T2	T3
IBW (g)	15.5 ± 0.02	15.5 ± 0.01	15.5 ± 0.03	15.5 ± 0.01	0.441
FBW (g)	37.3 ± 0.35 a	40.9 ± 0.27 c	39.7 ± 0.14 b	38.0 ± 0.08 a	0.001
SR (%)	98.3 ± 1.67	98.3 ± 1.67	100.0 ± 0.00	98.3 ± 1.67	0.802
WG (g/fish)	21.8 ± 0.34 a	25.4 ± 0.28 c	24.2 ± 0.13 b	22.5 ± 0.08 a	0.001
SGR (%/day)	1.79 ± 0.019 a	1.98 ± 0.015 c	1.92 ± 0.007 b	1.83 ± 0.004 a	0.001
CF	1.70 ± 0.036	1.74 ± 0.033	1.72 ± 0.021	1.72 ± 0.011	0.695
VSI (%)	10.45 ± 0.101	10.48 ± 0.202	10.50 ± 0.35	10.51 ± 0.096	0.988
HSI (%)	2.69 ± 0.044	2.72 ± 0.010	2.73 ± 0.073	2.71 ± 0.026	0.911
FC (g/fish)	24.0 ± 0.64 a	27.1 ± 0.80 b	25.7 ± 0.19 b	24.4 ± 0.31 a	0.008
FE	0.92 ± 0.004 a	0.95 ± 0.004 b	0.94 ± 0.002 b	0.94 ± 0.004 a	0.046
PER	1.89 ± 0.024 a	1.96 ± 0.040 b	1.96 ± 0.004 b	1.92 ± 0.025 a	0.011

Values (means of triplicates ± SE) in the same row sharing different superscript letters are significantly different (p < 0.05). Abbreviations: IBW, initial body weight; FBW, final body weight; SR, survival; WG, weight gain; SGR, specific growth rate; CF, condition factor; VSI, viscerosomatic index; HSI, hematosomatic index; FC, feed consumption; FE, feed efficiency; PER, protein efficiency ratio.

. After the 8-week feeding trial, SR did not differ among the treatments (p > 0.05). However, final body weight (FBW), WG, and SGR in the T1 and T2 groups were significantly (p < 0.05) enhanced compared with those in the T0 and T3 groups. Furthermore, the FC, FE, and PER in the T1 and T2 groups significantly (p < 0.05) improved compared with those in the T0 and T3 groups. No significant differences in CF, VSI, and HSI were observed among all groups.

3.2. Proximate Composition of Whole-Body and Plasma Biochemical Parameters

Table 3. Proximate composition (%, wet weight basis) and blood biochemical parameters of juvenile rockfish subjected to different YJB-supplemented diet feeding strategies.

	Experimental Diets				p-Value
	T0	T1	T2	T3
Moisture (%)	70.0 ± 0.13 a	69.8 ± 0.21 a	70.6 ± 0.21 a	69.9 ± 0.12 a	0.059
Crude protein (%)	14.8 ± 0.12 a	14.9 ± 0.15 a	15.0 ± 0.26 a	15.1 ± 0.41 a	0.797
Crude lipid (%)	8.4 ± 0.12 a	8.3 ± 0.12 a	8.6 ± 0.36 a	8.5 ± 0.06 a	0.746
Ash (%)	4.2 ± 0.17 a	4.2 ± 0.03 a	4.1 ± 0.17 a	4.1 ± 0.12 a	0.896
AST (U/L)	38.7 ± 3.18 a	34.3 ± 4.33 a	36.3 ± 3.53 a	32.3 ± 7.42 a	0.821
ALT (U/L)	22.7 ± 2.85 a	23.7 ± 2.40 a	24.0 ± 0.58 a	26.3 ± 0.67 a	0.601
T-CHO (mg/dL)	171.7 ± 3.93 a	173.0 ± 4.62 a	179.7 ± 7.62 a	172.0 ± 3.79 a	0.681
TP (g/dL)	4.2 ± 0.32 a	4.3 ± 0.41 a	4.9 ± 0.26 a	4.2 ± 0.35 a	0.421
GLU (mg/dL)	113.7 ± 5.36 a	111.0 ± 5.00 a	110.3 ± 3.53 a	117.0 ± 4.93 a	0.753

Values (means of triplicates ± SE) in the same row sharing different superscript letters are significantly different (p < 0.05). Abbreviations: AST, aspartate aminotransferase; ATL, alanine aminotransferase; T-CHO, total cholesterol; TP, total protein; GLU, glucose.

presents the effects of different feeding strategies regarding the dietary supplementation of YJB on the chemical whole-body composition and plasma biochemical indices of the fish. No significant differences in the moisture, crude protein, crude lipid, ash, AST, ALT, T-CHO, TP, and GLU content of the fish were detected between treatments (p > 0.05).

3.3. Lysozyme and Antioxidant Enzyme Activities

The lysozyme, SOD, and CAT activities and GSH content of the rockfish under each treatment are presented in

Table 4. Serum lysozyme activity and antioxidant status of juvenile rockfish under different YJB-supplemented diet feeding regimens.

	Experimental Diets				p-Value
	T0	T1	T2	T3
Lysozyme (U/mL)	1.34 ± 0.019 a	1.63 ± 0.022 c	1.47 ± 0.010 b	1.49 ± 0.046 b	0.000
SOD (U/mL)	1.50 ± 0.065 a	2.43 ± 0.065 c	2.38 ± 0.056 c	1.68 ± 0.035 b	0.000
CAT (nmol/min/mL)	315.4 ± 3.26 a	342.6 ± 3.36 b	335.4 ± 4.21 b	334.4 ± 3.84 b	0.004
GSH (µM)	2.39 ± 0.342 a	5.82 ± 0.642 c	5.73 ± 0.690 c	3.73 ± 0.316 b	0.004

Values (means of triplicates ± SE) in the same row sharing different superscript letters are significantly different (p < 0.05). Abbreviations: SOD, superoxide dismutase; CAT, catalase; GSH, glutathione.

. The serum lysozyme activity in the T1 group, followed by that in the T2 and T3 groups, was significantly higher (p < 0.05). The plasma SOD and GSH in the T1 and T2 groups, followed by those in the T3 group, were significantly (p < 0.05) higher than those in the T0 group. Among the parameters assessed in this study, lysozyme, SOD, and GSH were lowest in the T0 group (p < 0.05). CAT activity in the T1, T2, and T3 groups significantly (p < 0.05) increased compared with that in the control (T0) group, and there were no differences among different YJB feeding regimens.

3.4. Intestinal Digestive Enzyme Activities

The effects of different feeding strategies regarding the dietary supplementation of YJB on the activities of intestinal digestive enzymes, including amylase, trypsin, and lipase, are presented in

Table 5. Digestive enzyme activities of juvenile rockfish under different feeding regimens with respect to the YJB-supplemented diet.

	Experimental Diets				p-Value
	T0	T1	T2	T3
Amylase (U/L)	375.1 ± 13.0 a	702.2 ± 17.8 b	693.9 ± 16.8 b	423.8 ± 16.0 a	0.000
Trypsin (U/L)	33.0 ± 3.26 a	55.8 ± 2.30 b	54.0 ± 3.08 b	39.8 ± 3.76 a	0.002
Lipase (U/L)	48.2 ± 2.51 a	76.2 ± 9.08 b	67.9 ± 2.29 b	50.3 ± 4.23 a	0.015

Values (means of triplicates ± SE) in the same row sharing different superscript letters are significantly different (p < 0.05).

. All digestive enzyme activities were significantly (p < 0.05) higher in the T1 and T2 groups than in the T0 and T3 groups.

3.5. Challenge Test

Figure 1 shows the survival rates of the fish artificially infected with S. iniae 12 days after infection. The survival rates of the fish fed YJB and challenged with S. iniae were significantly (p < 0.05) higher than those of the control group. The fish fed a continuous supply of YJB (T2 group) showed the highest survival rates among all the tested groups.

4. Discussion

The effect of the T1 (feeding the fish YJB continuously) and T2 (feeding them YJB for 1 day and the basal diet on the following day) treatments on growth (FBW, WG, and SGR), feed utilization (FC, FE, and PER), and digestive enzyme activity showed a significant difference from that of the T0 treatment (feeding the fish a basal diet continuously). FOSs, a prebiotic present in YJB, are implicated in the digestion, absorption, and metabolism of essential nutrients in aquatic animals [35,36,37,38,39]. The growth performance of blunt snout bream (Megalobrama amblycephala) administered 4 g kg⁻¹ of FOSs discontinuously was better than that of a control group fed a basal diet continuously but was not significantly different from that of fish fed 4 g kg⁻¹ of FOSs continuously [39]. Dimitroglou et al. [40] and Zhang et al. [39] indicated that the growth performance of fish is directly related to intestinal enzyme activities and the length and quantity of the intestinal villus, which may aid digestive and absorption processes in the intestine. Bai et al. [41] showed that white shrimp (Litopenaeus vannamei) fed dietary β-glucan for 2 days followed by a basal diet for 5 days exhibited the highest specific growth rate. However, the T3 treatment (i.e., feeding them YJB for 1 day and the basal diet for the next 2 days) showed no significant difference from the T0 treatment (i.e., feeding them the basal diet continuously). Additionally, Merrifield et al. [42] and Tachibana et al. [33] demonstrated that a feeding strategy involving the provision of feed additives to fish may induce distinct growth responses. The differences in growth performance depending on the feeding strategy used for feed additives can be attributed to several factors, including the duration of the additive’s effect, dietary additive concentrations, and the physiological requirements of fish species. However, given the lack of relevant studies, the exact mechanism underlying this process is unclear and requires additional investigation.

Digestive enzymes are a combination of enzymes found in the intestines of animals and are responsible for breaking down macromolecules into their smaller forms to increase the metabolism of macromolecules [43]. Trypsin, lipase, and amylase are the major digestive enzymes produced by fish for feed digestion and absorption. Additionally, the body’s metabolism may increase if the levels of these enzymes rise [44]. A previous study demonstrated that, in fish, YJB may stimulate digestive enzyme activity to stimulate appetite [28]. In particular, FOSs in YJB have been demonstrated to be highly helpful in boosting digestive enzyme activity in fish [38,45,46,47]. In addition, the proteases, amylases, and lipases released by FOSs may complement the shortage of digestive enzymes in fish, enhance the digested product content in chyme, and activate intestinal chemoreceptors and, thus, induce the production of endogenous digestive enzymes [38]. In this work, the digestive enzyme activity in the T2 and T3 groups was significantly enhanced relative to that in the T0 group, thus indicating that the administration of YJB at 1-day intervals increased growth and feed utilization by enhancing the activity of intestinal digestive enzymes in the juvenile rockfish.

In this study, different YJB feeding regimens did not appear to affect the body compositions (moisture, crude protein, crude lipid, and ash) of the juvenile rockfish. These results indicated that none of the YJB feeding regimens appeared to inhibit nutrient absorption. Similar to this study, previous works showed that the type and dosage of the supplementation of yacon products, including YJB, had no effect on the body compositions of fish [28,29,48].

Blood biochemical parameters can be employed as physiological biomarkers of the improvements in fish health induced by the inclusion of functional feed additives [49,50,51]. However, in this study, the juvenile rockfish subjected to different feeding YJB regimens had normal plasma biochemical parameters. These results revealed that the fish were in good health and that YJB had no negative effects on the blood biochemical indices of the juvenile black rockfish [28,29].

Lysozyme, an essential component of the body’s nonspecific immunity, can dissolve Gram-positive bacteria and kill Gram-negative bacteria whose cell walls have been eliminated by the body [52,53]. In this study, the serum lysozyme activity of the rockfish under all YJB feeding regimens was considerably higher than that of the fish fed the basal diet, hence increasing resistance against S. iniae bacterial infection. Lee et al. [29] and Oh et al. [28] reported that supplementing the feed of juvenile black rockfish with YJB enhanced serum lysozyme activity and then decreased mortality rates after a challenge with Gram-positive (Vibrio anguillarum) and Gram-negative (S. iniae) bacteria. In addition, fish treated with FOS are well known to show enhanced immunity [54]. Zhang et al. [39] demonstrated that dietary FOSs exerted beneficial effects on the nonspecific immune responses of snout bream, as indicated by the significantly higher levels of immunological parameters (including serum lysozyme) in FOS-fed fish. These results are comparable with the findings described in this study, demonstrating that a suitable YJB feeding frequency might boost the nonspecific immunity of rockfish.

SOD is an essential antioxidant enzyme in the body. It can remove O²⁻ free radicals and transform O²⁻ into H₂O₂. It is destroyed by CAT and GSH, hence maintaining a lower state of equilibrium for free radicals to prevent oxidative damage to functional macromolecules [55,56]. This study revealed that, consistent with the nonspecific response (lysozyme activity), plasma SOD, CAT, and GSH activities were all enhanced by the application of the YJB feeding regimen. These results indicated that all the YJB feeding strategies could enhance the antioxidant enzyme activity of the fish. Similarly, FOSs might enhance the antioxidant capacity of juvenile blunt snout bream [39], as supported by the fact that antioxidant enzymes can scavenge reactive oxygen species (ROS) and lipid peroxidation products, thereby protecting cells and tissues from oxidative damage [55].

Cumulative mortality is a crucial measure used to assess the health of cultured organisms and the influence of immune stimulants [57]. At the end of this experiment, the cumulative mortality of the juvenile rockfish in all the groups fed YJB was significantly lower than that in the control group, thus indicating that, when given at a reasonable feeding frequency, YJB acts as an activator that effectively improves the cellular and humoral immune functions of fish. This improvement, in turn, results in significantly increased resistance against S. iniae infection. Other studies ascribed this enhanced disease resistance to the ability of FOSs to improve host defense while also stimulating the immune system by increasing immunoglobulin M production and cytokine regulation [58]. These results may indicate that the different strategies of incorporating YJB into feed enhance the non-specific immune response (lysozyme and antioxidant enzyme activities), a result that may be related to the increased resistance of juvenile black rockfish to pathogenic S. iniae.

5. Conclusions

In conclusion, YJB offered every 2 days ameliorated the nonspecific immune response and antioxidant status of juvenile black rockfish. YJB should be supplied at day-to-day intervals to improve the growth performance, digestive enzyme activity, and blood health of fish. Feeding the fish the basal diet for 1 day followed by feeding them the 2.5 g/kg YJB diet for 1 day was the most suitable feeding regimen for juvenile black rockfish culture given its economic cost-saving effect.